About the Yadavalli Lab
Bacterial stress responses allow cells to sense, adapt, and survive against various stressors in their environment. While the stress response networks are necessary for bacterial defense and survival, they can also contribute to the rise of antimicrobial resistance. Antimicrobial resistance is an alarming problem of the present and future. Understanding the biochemical and regulatory pathways that underlie this resistance is of utmost importance to tackle the growing threat of untreatable multidrug-resistant bacterial infections. Research in the Yadavalli lab is focused on two emerging themes in bacterial gene regulation:
(i) Small regulatory proteins – proteins with less than 50 amino acids that directly translated from short open reading frames.
(ii) Epitranscriptomic proteins – epitranscriptomic regulators that connect RNA modifications and translation to metabolism and stress response.
BIOLOGY OF SMALL PROTEINS
In the past decade, numerous small proteins have been discovered in both eukaryotes (<100 amino acids) and prokaryotes (<50 amino acids). It is becoming increasingly clear that this class of proteins is important for the regulation of cell physiology under stress conditions. About one-third of these newly discovered proteins are predicted to be membrane-bound, where they can modulate signal transduction, transport, and/or other enzymatic activities (Yadavalli & Yuan, 2021). Uncovering the identities and functions of small proteins will expand our understanding of this under-explored class of regulatory molecules in the cell. We have a very limited understanding of most aspects of small protein biology. To expand our understanding of the mechanistic and physiological roles of small proteins, we are developing biochemical tools to a) identify cellular targets of these small proteins, and b) study the roles of small protein regulators in stress response to specific signals. We recently published an updated ribosome-profiling approach (Cope*, Vellappan* et al., 2021), which we are currently using to study the condition-specific expression of small proteins. We aim to systematically characterize each of these proteins to identify their functions in the cell. An example of such functional characterization of a small protein comes from our recent study of a small membrane peptide MgrB, an inhibitor of PhoQ sensor kinase. We analyzed the molecular details of MgrB-PhoQ interactions using a combination of biochemical and biophysical approaches including beta-galactosidase reporter gene assays, single-cell fluorescence measurements, bacterial two-hybrid tests, as well as fluorescence resonance energy transfer (Yadavalli et al., 2020). We identified a group of specific functionally important residues in the transmembrane as well as periplasmic regions of this protein. Interestingly, despite the small length of MgrB, a majority of amino acids are dispensable based on alanine-scanning mutagenesis. In addition to the aforementioned techniques, we are using high-throughput transcriptomic and proteomic tools to decipher the roles of small protein regulators.
ANTIMICROBIAL PEPTIDE STRESS RESPONSE MEDIATED BY AN EPITRANSCRIPTOMIC PROTEIN
In our previous work, we found that treating E. coli with sublethal concentrations of an antimicrobial peptide (C18G) causes cells to filament (Yadavalli et al., 2016). This filamentation is dependent on the PhoQ/PhoP two-component signaling network, important for survival in response to signals such as low magnesium, acidic pH, osmotic upshift, and the presence of cationic antimicrobial peptides, and regulates virulence in E. coli, Salmonella, and related bacteria. This work demonstrates that the block in cell division is not due to cell wall/membrane damage induced by the antimicrobial peptide, but instead the result of a high stimulus through this two-component system. Filamentation is mediated by an enzyme, QueE, which participates in the biosynthesis of a tRNA modification called queuosine (Q). We are currently investigating the details of the mechanism of cell division inhibition by QueE in response to antimicrobial peptide stress.